Background: Root hair culture is a valuable system to produce recombinant proteins in planta. Antimicrobial peptides (AMPs) are vital parts of the innate immune response found in almost all forms of life. Precise target activity and limited toxicity towards mammalian cells make them suitable candidate molecules to combat evolving drug-resistant microorganisms. The aim of the present study was to produce a Dermaseptin B1 recombinant antimicrobial peptide in Nicotiana tabacum root hair and assess the antibacterial activity of the protein extract from transgenic root hairs.Materials and Methods: A Dermaseptin B1 encoding gene sequence was C-terminally fused to a Chitin Binding Domain (CBD) encoding sequence and cloned in a plant binary vector used for Agrobacteriumrhizogenes-mediated transformation to generate root hairs in tobacco. Transgenic root hairs were produced, and protein extracts were used to assess antimicrobial activity against a number of microbes.Results: PCR and RT-PCR analysis confirmed the integration of the Dermaseptin B1 gene in a root hair cell genome and the presence of Dermaseptin B1 mRNA transcripts, respectively. Recombinant protein had a significant (P<0.05) antibacterial effect towards gram-positive and gram-negative bacteria.Conclusion: Dermaseptine B1 recombinant peptide was successfully produced in tobacco root hair cells and its antibacterial effects was confirmed. These results suggest that the recombinant protein may have a therapeutic effect to control bacterial pathogens. It can be concluded that root hair cells can be used to produce and purify valuable recombinant proteins with pharmaceutical applications.